A novel high performing multiplex immunoassay Multi-HTLV for serological confirmation and typing of HTLV infections.

BACKGROUND: Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains. METHODOLOGY/PRINCIPAL FINDINGS: The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results. CONCLUSIONS/SIGNIFICANCE: The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Accurate Confirmatory Diagnosis of HTLV-1/2 Infection.

Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 (n = 125), HTLV-2 (n = 19), and coinfected with HIV (n = 82), and compared with real-time polymerase chain reaction (qPCR) and PCR-restriction fragment length polymorphism (RFLP). The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8-85.5%) was slightly superior to qPCR (95% CI 69.5-81.1%) and similar to PCR-RFLP (95% CI 79.5-89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2-93.4%) than for HTLV-2 (95% CI 43.2-70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.

[Detection of Human T lymphotropic virus 1 (HTLV-1) Cosmopolitan subtype Transcontinental subgroup (Aa) and HTLV-2 subtype b in blood donors of Corrientes]. / Hallazgo del virus linfotrópico T humano 1 (HTLV-1) subtipo Cosmopolita subgrupo Transcontinental (Aa) y del HTLV-2 subtipo b en donantes de sangre de Corrientes.

A molecular epidemiological study was conducted in a population of 9422 blood donors in the province of Corrientes, Northeastern Argentina, to determine the prevalence of Human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2), the phylogenetic identification of HTLV-1 and 2 subtypes/subgroups and perform a mutation analysis. Based on the results obtained, it was shown that both HTLV-1 and HTLV-2 are circulating in a low-risk population of Corrientes, although with a similar prevalence to that of non-endemic areas. Phylogenetic studies identified the HTLV-1 Cosmopolitan subtype Transcontinental subgroup (Aa), and the HTLV-2 subtype b. Infected donors reported neither a history of risk factors such as transfusions, intravenous drug use, nor risky or HTLV-1/2 seropositive sexual partners. These results suggest that these viruses were transmitted from mother to child, possibly from generation to generation, and that these strains were introduced into the Caucasian population of this region from ancestors originating from endemic areas of the country either from or through contact with individuals from other countries years ago. Our results demonstrate for the first time the presence of HTLV-1 and HTLV-2 in the province of Corrientes. Moreover, although the province can be considered a non-endemic area, the need to include these retroviruses in a national Public Health program is highlighted, in order to have qualified professionals duly trained to make their diagnosis and provide the necessary information in relation to primary care and patient follow-up.

HTLV-1 and -2 in a first-time blood donor population in Northeastern Brazil: Prevalence, molecular characterization, and evidence of intrafamilial transmission.

Independent epidemiology for respective human T-cell lymphotropic virus (HTLV) types 1 and 2 is little known in blood donors in Brazil, where screening for HTLV-1/2 is mandatory at blood banks, but no testing to confirm/differentiate these viruses. Therefore, this study aims to assess the prevalence of HTLV-1 and -2 in a first-time blood donor population in Northeastern Brazil and to carry out molecular characterization of respective isolates. A cross-sectional study was conducted at the State Blood Bank in Piauí. Samples were screened for anti-HTLV-1/2 by enzyme immunoassay, and reactive samples were confirmed using a line immunoassay and polymerase chain reaction (PCR). Of 37 306 blood donors, 47 were anti-HTLV-1/2 reactive by enzyme immunoassay. After confirmed by line immunoassay, 22 were positive for HTLV-1 (0.59 per 1000; 95% CI: 0.38-0.87), 14 were positive for HTLV-2 (0.37 per 1000; 95% CI: 0.21-0.61), 1 was indeterminate, and the remaining donors were negative. The HTLV-1 infection was also confirmed by PCR in all anti-HTLV-1-positive samples, and sequencing classified these isolates as belonging to the Transcontinental (A) subgroup of the Cosmopolitan (1a) subtype. Of 14 anti-HTLV-2-positive samples, 11 were also PCR positive, which belonged to subtype a (HTLV-2a/c). In addition, 38 family members of 5 HTLV-1- and 3 HTLV-2-infected donors were analyzed. Familial transmission of HTLV-1 and -2 was evidenced in 3 families. In conclusion, in Northeastern Brazil, where HTLV-1 and -2 are endemic, counseling blood donor candidates and their families might play a key role in limiting the spread of these viruses.

Identification of rare HIV-1 Group N, HBV AE, and HTLV-3 strains in rural South Cameroon.

Surveillance of emerging viral variants is critical to ensuring that blood screening and diagnostic tests detect all infections regardless of strain or geographic location. In this study, we conducted serological and molecular surveillance to monitor the prevalence and diversity of HIV, HBV, and HTLV in South Cameroon. The prevalence of HIV was 8.53%, HBV was 10.45%, and HTLV was 1.04% amongst study participants. Molecular characterization of 555 HIV-1 specimens identified incredible diversity, including 7 subtypes, 12 CRFs, 6 unclassified, 24 Group O and 2 Group N infections. Amongst 401 HBV sequences were found a rare HBV AE recombinant and two emerging sub-genotype A strains. In addition to HTLV-1 and HTLV-2 strains, sequencing confirmed the fifth known HTLV-3 infection to date. Continued HIV/HBV/HTLV surveillance and vigilance for newly emerging strains in South Cameroon will be essential to ensure diagnostic tests and research stay a step ahead of these rapidly evolving viruses.

Dual Simian Foamy Virus/Human Immunodeficiency Virus Type 1 Infections in Persons from Côte d'Ivoire.

Zoonotic transmission of simian retroviruses in West-Central Africa occurring in primate hunters has resulted in pandemic spread of human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). While simian foamy virus (SFV) and simian T- lymphotropic virus (STLV)-like infection were reported in healthy persons exposed to nonhuman primates (NHPs) in West-Central Africa, less is known about the distribution of these viruses in Western Africa and in hospitalized populations. We serologically screened for SFV and STLV infection using 1,529 specimens collected between 1985 and 1997 from Côte d'Ivoire patients with high HIV prevalence. PCR amplification and analysis of SFV, STLV, and HIV/SIV sequences from PBMCs was used to investigate possible simian origin of infection. We confirmed SFV antibodies in three persons (0.2%), two of whom were HIV-1-infected. SFV polymerase (pol) and LTR sequences were detected in PBMC DNA available for one HIV-infected person. Phylogenetic comparisons with new SFV sequences from African guenons showed infection likely originated from a Chlorocebus sabaeus monkey endemic to Côte d'Ivoire. 4.6% of persons were HTLV seropositive and PCR testing of PBMCs from 15 HTLV seroreactive persons identified nine with HTLV-1 and one with HTLV-2 LTR sequences. Phylogenetic analysis showed that two persons had STLV-1-like infections, seven were HTLV-1, and one was an HTLV-2 infection. 310/858 (53%), 8/858 (0.93%), and 18/858 (2.1%) were HIV-1, HIV-2, and HIV-positive but undifferentiated by serology, respectively. No SIV sequences were found in persons with HIV-2 antibodies (n = 1) or with undifferentiated HIV results (n = 7). We document SFV, STLV-1-like, and dual SFV/HIV infection in Côte d'Ivoire expanding the geographic range for zoonotic simian retrovirus transmission to West Africa. These findings highlight the need to define the public health consequences of these infections. Studying dual HIV-1/SFV infections in immunocompromised populations may provide a new opportunity to better understand SFV pathogenicity and transmissibility in humans.

Molecular characterization of the human T cell lymphotropic virus type 2 long terminal repeat region: A discussion about possible influences at viral gene expression.

This study aimed to identify nucleotide signatures in the promoter region of human T cell lymphotropic virus type 2 (HTLV-2) isolated from infected individuals from Salvador, Brazil and in sequences from the GenBank database. DNA samples from HTLV-2-infected individuals were submitted to nested polymerase chain reaction (PCR) and sequencing, and molecular analyses were performed using bioinformatics tools. The phylogeny of HTLV-2 strains isolated from patients from Salvador reveals that all sequences were subtype c. One hundred and fifty-one sequences from GenBank were selected, among which 30 belong to subtype a, 88 to subtype b, 32 to subtype c, and one to subtype d. Subtype-specific signatures were identified as well as mutations resulting in loss or gain of motifs important to transcription regulation. The subtypes a and b have two E box motifs, while subtypes c and d have only one. These polymorphisms may impact viral fitness and infection outcome and should be more closely investigated.

Molecular epidemiology of HIV, HBV, HCV, and HTLV-1/2 in drug abuser inmates in central Javan prisons, Indonesia.

INTRODUCTION: This study was conducted to determine the current molecular prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and human T lymphotropic virus-1/2 (HTLV-1/2) circulating among drug abuser inmates incarcerated in prisons located in Central Java, Indonesia. METHODOLOGY: Socio-epidemiological data and blood specimens were collected from 375 drug abuser inmates in four prisons. The blood samples were analyzed with serological and molecular testing for HIV, HBV, HCV, HDV, and HTLV-1/2. RESULTS: The seroprevalence of HIV, HBsAg, HCV, HDV, and HTLV-1/2 in drug abuser inmates was 4.8% (18/375), 3.2% (12/375), 34.1% (128/375), 0% (0/375), and 3.7% (14/375), respectively. No co-infections of HIV and HBV were found. Co-infections of HIV/HCV, HIV/HTLV-1/2, HBV/HCV, HBV/HTLV-1/2, and HCV/HTLV-1/2 were prevalent at rates of 4% (15/375), 1.3% (5/375), 1.1% (4/375), 0.3% (1/375), and 2.1% (8/375), respectively. The HIV/HCV co-infection rate was significantly higher in injection drug users (IDUs) compared to non-IDUs. Triple co-infection of HIV/HCV/HTLV-1/2 was found only in three IDUs (0.8%). HIV CRF01_AE was found to be circulating in the inmates. HBV genotype B3 predominated, followed by C1. Subtypes adw and adr were found. HCV genotype 1a predominated among HCV-infected inmates, followed by 1c, 3k, 3a, 4a, and 1b. All HTLV-1 isolates shared 100% homology with HTLV-1 isolated in Japan, while all of the HTLV-2 isolates were subtype 2a. CONCLUSION: Drug abuser inmates in prisons may offer a unique community to bridge prevention and control of human blood-borne virus infection to the general community.

Human T cell lymphotropic virus type 2a strains among HIV type 1-coinfected patients from Brazil have originated mostly from Brazilian Amerindians.

The human T cell lymphotropic virus type 2 (HTLV-2) is found mainly in Amerindians and in intravenous drug users (IDUs) from urban areas of the United States, Europe, and Latin America. Worldwide, HTLV-2a and HTLV-2b subtypes are the most prevalent. Phylogenetic analysis of HTLV-2 isolates from Brazil showed the HTLV-2a subtype, variant -2c, which spread from Indians to the general population and IDUs. The present study searched for the types of HTLV-2 that predominate among HIV-1-coinfected patients from southern and southeastern Brazil. Molecular characterization of the LTR, env, and tax regions of 38 isolates confirmed the HTLV-2c variant in 37 patients, and one HTLV-2b in a patient from Paraguay. Phylogenetic analysis of sequences showed different clades of HTLV-2 associated with risk factors and geographic region. These clades could represent different routes of virus transmission and/or little diverse evolutionary rates of virus. Taking into account the results obtained in the present study and the lack of the prototypic North American HTLV-2a strain and HTLV-2b subtypes commonly detected among HIV-coinfected individuals worldwide, we could speculate on the introduction of Brazilian HTLV-2 strains in such populations before the introduction of HIV.

Stable human T-cell lymphotropic virus type 1 (HTLV-1) subtype a/subgroup a endemicity in Amerindians from Northwest Argentina: a health problem to be resolved.

Jujuy province, in Northwest Argentina, is known to be endemic for HTLV-1 infection. Moreover, foci of HTLV-1 associated pathologies have also been described in this region. To gain an insight into the current situation of HTLV-1/2 in this endemic area, a seroprevalence and phylogenetic study was performed among a Kolla community from Abra Pampa city and surroundings. Out of 112 individuals, 11 (9.8%) were confirmed as HTLV-1 positive and no HTLV-2 infection was detected. The phylogenetic analysis of the LTR region showed that all the HTLV-1 sequences belonged to the Cosmopolitan subtype a/transcontinental subgroup A, and were closely related to reference sequences from Peru, Argentina, and the South of Brazil (P = 0.82). Considering the cultural and historical features of this community and in spite of the mandatory detection of anti-HTLV-1/2 antibodies in blood banks since 2005, it would be important to implement new public health measures focused on decreasing HTLV-1 transmission in this endemic area.

Development and validation of a multiplex real-time PCR assay for simultaneous genotyping and human T-lymphotropic virus type 1, 2, and 3 proviral load determination.

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10(5) to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r(2) = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.

Serologic and molecular typing of human T-lymphotropic virus among blood donors in Maputo City, Mozambique.

BACKGROUND: Screening for human T-lymphotropic virus-1/2 (HTLV-1/2) infection is not performed in blood banks in Mozambique. The aim was to determine the prevalence of HTLV-1/2 among blood donors of the Maputo Central Hospital Blood Bank and measure the coinfection rate of HTLV-1/2 with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and syphilis. STUDY DESIGN AND METHODS: A total of 2019 consecutive blood donors were screened for HTLV-1/2 antibodies, HIV-1/2 antibodies, hepatitis B surface antigen (HBsAg), and rapid plasma reagin (RPR) for syphilis. Specimens reactive on a first HTLV-1/2 enzyme immunoassay (EIA) were retested using a second EIA. Specimens that were dually reactive on both EIAs were further tested using Western blot (WB) and real-time polymerase chain reaction (PCR). RESULTS: All 18 dually reactive specimens (0.89%; 95% confidence interval, 0.48%-1.30%) were positive for the presence of HTLV-1 by WB and real-time PCR. HTLV-2 was not detected. The prevalences of anti-HIV, HBsAg, and reactivity in the RPR test were 5.72, 6.01, and 0.98 percent, respectively. There was no significant association between HTLV-1 infection and demographic variables (age and sex) or serologic markers (HIV, HBsAg, and RPR). For the 17 HTLV-1-positive donors for whom serologic data for HIV, HBsAg, and syphilis RPR were available, 2 showed coinfection with HIV and 1 with HBV. CONCLUSION: Compared to other infectious agents, HTLV-1 is present at relatively low levels among blood donors in Mozambique. Cost and logistics will present as major challenges for introducing HTLV-1/2 screening in blood banks. In blood banks in Southern Africa where EIA testing is possible, a sequential algorithm of two EIAs may be a cost-efficient option for HTLV-1/2 screening.

The human HTLV-3 and HTLV-4 retroviruses: new members of the HTLV family.

Human T cell leukemia/lymphoma virus Type 1 and 2 (HTLV-1 and HTLV-2), together with their simian counterparts (STLV-1, STLV-2), belong to the Primate T lymphotropic viruses group (PTLV). HTLV-1 infects 15 to 20million people worldwide, while STLV-1 is endemic in a number of simian or ape species living in Africa or Asia. The high percentage of homologies between HTLV-1 and STLV-1 strains, led to the demonstration that most HTLV-1 subtypes arose from interspecies transmission between monkeys and humans. STLV-3 viruses belong to the third PTLV type and are equally divergent from HTLV-1 than from HTLV-2. They are endemic in several monkey species that live in West, Central, and East Africa. In 2005, we and others reported the discovery of the human homolog (HTLV-3) of STLV-3 in two asymptomatic inhabitants from South Cameroon whose sera exhibited HTLV indeterminate serologies. More recently, we reported a third case of HTLV-3 infection in Cameroon suggesting that this virus is not rare in the human population living in Central Africa. Together with STLV-3, these three human viral strains belong therefore to the PTLV-3 type. A fourth HTLV type (HTLV-4) was also discovered in the same geographical area. Current studies are aimed at determining the prevalence, distribution and modes of transmission of these viruses as well as their possible association with human diseases. Furthermore, molecular characterization of their viral transactivator Tax is ongoing in order to look for possible oncogenic properties.

New insights into prevalence, genetic diversity, and proviral load of human T-cell leukemia virus types 1 and 2 in pregnant women in Gabon in equatorial central Africa.

Human T-cell leukemia virus type 1 (HTLV-1) is highly endemic in areas of central Africa; mother-to-child transmission and sexual transmission are considered to be the predominant routes. To determine the prevalence and subtypes of HTLV-1/2 in pregnant women in Gabon, we conducted an epidemiological survey in the five main cities of the country. In 907 samples, the HTLV-1 seroprevalence was 2.1%, which is lower than that previously reported. Only one case of HTLV-2 infection was found. The HTLV-1 seroprevalence increased with age and differed between regions (P

Human T-cell lymphotropic virus types I and II (HTLV-I and -II) infection among seroindeterminate cases in Argentina.

Human T-cell lymphotropic virus (HTLV) seroindeterminate cases have been reported among blood donors (BD) and in at-risk populations worldwide, including Argentina. The objective of the present work was to study the presence of HTLV-I/II infection and its association to specific Western blot (WB) patterns among healthy BD and at-risk populations in Argentina. We analyzed 83 HTLV-I/II seroindeterminate WB cases diagnosed among BD (n = 49) and in different at-risk populations (n = 34) for human retroviruses infections. Multiple indeterminate WB patterns were observed. Out of the total, 13.2% (11/83) of the cases were found to be HTLV-I/II positive by nested-PCR (n-PCR), including 13.2% (11/83) HTLV-I and 2.4% (2/83) presenting HTLV-I and -II co-infection. Most of their serological profiles showed reactivity to gag or env codified proteins. Two samples amplified only one of the six analyzed genes (1 HTLV-I pol gene and 1 HTLV-II tax gene). There was no association between the presence of Trypanosoma cruzi infection and an HTLV-I/II indeterminate WB pattern (only 3 of the 83 samples were positive for T. cruzi antibodies). In conclusion, the majority of HTLV-seroindeterminate WB donors lacked HTLV provirus and was thus considered uninfected. However, when seroreactivity to Env and Gag proteins are observed on the WB and especially in at-risk populations, HTLV infection should be suspected; such individuals should be followed-up and retested.

Molecular characterization of human T-cell lymphotropic virus type 2 (HTLV-II) from people living in urban areas of Sao Paulo city: evidence of multiple subtypes circulation.

BACKGROUND: In Brazil, human T-cell lymphotropic virus type I and type II (HTLV-I and HTLV-II) are co-circulating and possess approximately 65% homology, which results in high cross-reactivity in serological tests. Based on the detection of EIA and Western blot (WB) tests, HTLV serodiagnosis yields indeterminate results in high-risk population, with the true determination of HTLV-II prevalence requiring a combined serological and molecular analysis. Molecular analysis of HTLV-II isolates has shown the existence of four distinct subtypes: IIa, IIb, IIc, and IId. The aim of this study was to evaluate the routine EIA and WB used in Sao Paulo city, as well as molecular methods for confirmation of infection and HTLV-II subtype distribution. RESULTS: Two hundred ninety-three individuals, who were enrolled in the HTLV out-clinic in Sao Paulo city, Brazil, between July 1997 and May 2003, were tested by EIAs, and positive sera 232 (79%) reactive by one of the tests. When these sera were tested by WB revealed 134 were HTLV-I, 28 HTLV-II, 4 HTLV-I/II, and 48 were indeterminate. Polymerase chain reaction (PCR) on the indeterminate group showed that 20 (42%) were HTLV-II and 28 were negative. From a total of 48 HTLV-II subjects with DNA available, restriction fragment length polymorphism (RFLP) of the env region revealed 47 HTLV-IIa and 1 HTLV-IIb. The phylogenetic analysis was performed on 23 samples, which identified 19 as subtype a, Brazilian subcluster, and 4 as subtype b. This is the first time HTLV-II subtype b has been described in Brazil. However, further studies, such as a complete nucleotide DNA sequencing, need to be done to confirm these findings.

Molecular evidence of HTLV-II subtype B among an urban population living in South Brazil.

Human T cell lymphotropic virus type II (HTLV-II) is a deltaretrovirus endemic in Indian populations living in Central and South America, among Pygmies tribes from Central Africa, and epidemic among injecting drug users (IDUs) in the United States, Europe, Southeast Asia, and South America. To date only the HTLV-IIa subtype has been demonstrated among Brazilians (Amazon basin Indians, blood donors, and IDUs). We analyzed HTLV-II isolates from 12 individuals living in the urban area of Porto Alegre, Southern Brazil, identified as seropositive for HTLVI/II in a blood donation. The HTLV-II long terminal repeat (LTR) region was sequenced and compared with nucleotide sequences of isolates HTLV-IIa (Mo), HTLV-IIb (NRA) prototypes. Phylogenetic analysis of the LTR region demonstrated that seven new isolates clustered together with American Indians HTLV-IIb isolates, and five new HTLV-IIa isolates clustered within the HTLV-IIa Brazilian subgroup, named the HTLV-IIc subtype. Both HTLV-IIa and IIb seem to be endemic in the urban area of Porto Alegre, South of Brazil, and could have reached this region via the Amazon basin and the Pacific Coast ancient human migratory pathways. To our knowledge this is the first study demonstrating the presence of HTLV-IIb among the urban population in Brazil.

Functional analysis of human T lymphotropic virus type 2 Tax proteins.

BACKGROUND: The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays. RESULTS: We found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis of the sequence of the two inactive Tax 2A proteins relative to Mo indicated that one had six amino acid changes and the other had one change in the central region of the protein. Mutations present at the amino and the extreme carboxy termini of Mo resulted in the loss of LTR but not NFkB activation whereas those occurring in the central region of the protein appeared to abolish transactivation of both promoters. Analysis of the transactivation phenotypes of Tax 1, Tax 2A Mo and Tax 2B containing mutations identified in the present study or previously characterised Tax mutations showed that domains required for LTR and NFkB activation are very similar but not identical in all three Tax proteins. CONCLUSION: Our results suggest that loss of activity of two Tax 2A proteins derived from different isolates is associated with multiple amino acid changes relative to Mo in domains required for the activation of the CREB or CREB and NFkB pathways and that these domains are very similar but not identical in Tax 2B and Tax 1. The loss of Tax function in 2A viruses may have implications for their biological and pathogenic properties.

Human T lymphotropic virus types I and II western blot seroindeterminate status and its association with exposure to prototype HTLV-I.

Human T lymphotropic virus types I and II (HTLV-I/II) Western blot (WB) seroindeterminate status, which is defined as an incomplete banding pattern of HTLV protein Gag (p19 or p24) or Env (GD21 or rgp46), is commonly observed. To investigate the significance of this finding, we examined HTLV-I/II serostatus and HTLV-I proviral load in 2 groups of individuals with WB seroindeterminate status. Low proviral loads were detected in 42% of patients with neurologic symptoms and 44% of voluntary blood donors. These data suggest that a subset of WB seroindeterminate individuals may be infected with prototype HTLV-I. To confirm this hypothesis, we evaluated HTLV-I/II serostatus and proviral load in prospectively collected specimens from 66 WB seronegative patients who had received HTLV-I-infected blood products by transfusion. Eight individuals developed WB seroindeterminate profiles after the transfusion. In addition, using a human leukocyte antigen type A*201-restricted HTLV-I Tax11-19 tetramer, we detected virus-specific CD8(+) T cells in peripheral blood mononuclear cells from WB seroindeterminate patients. These CD8(+) T cells were effective at targeting HTLV-I-infected cells. Collectively, the results suggest that HTLV-I/II WB seroindeterminate status may reflect a history of HTLV-I exposure. Our findings warrant further investigation of the possible clinical outcomes associated with WB seroindeterminate status.